In Vitro and In Vivo Evaluation of Titanium Surface Modification for Biological Aging by Electrolytic Reducing Ionic Water
Round 1
Reviewer 1 Report
This paper seems to have the potential to be published however some changes have to be made or discussed in order to be accepted.
Here summarized the few points:
1)The UV group always show better results in all test compared to Control and S-100. Therefore is it not clear why S-100 should ever be prefer to the UV in any future experiment or clinical trial. Please Clarify.
2)The UV group is used just in the in-vitro experiments and not the in-vivo although it shows better results than the other two groups; It should be used in the in-vitro model as well or an explanation for the exclusion should be made clear.
3)The experiments performed in the in-vivo model are limited and could be easily improved by adding more staining at support of what claimed. An osteoclastic staining would be interesting as well and would increase the significance of the content.
4)The abstract should be re-written to be more clear and easy to read. Minor rewriting would be also beneficial in the rest of the paper.
Author Response
1) The UV group always shows better results in all test compared to Control and S-100. Therefore is it not clear why S-100 should ever be preferred to the UV in any future experiment or clinical trial. Please Clarify.
A: Thank you for your comment. The UV treatment showed better results in our in vitro tests compared to S-100 treatment. However, S-100 treatment showed significantly high bioactivity compared to Control. Therefore, we think that S-100 is very useful for clinical setting because S-100 treatment which is inexpensive, requires no special equipment, can easily improve biological aging with a short processing time compare to the UV treatment.
2) The UV group is used just in the in vitro experiments and not the in vivo although it shows better results than the other two groups; It should be used in the in vitro model as well or an explanation for the exclusion should be made clear.
A: Many in vivo experiments with UV irradiation to improve biological aging have been reported. And the accretion of the new bone formation on the UV treated titanium surface have been widely known. Also in this in vitro test, the UV treatment showed good results in comparison with the S-100 treatment. Therefore, we dare thought that new bone formation in the UV treatment is better than S-100 treatment without intentionally conducting in vivo experiments in this study. Furthermore, as we received recommendations from our animal care committee to reduce the number of animals as much as possible according to the concept of 3R ("Replacement", "Reduction", "Refinement"), we excluded the UV treatment groups.
We added this information in discussion.
3) The experiments performed in the in vivo model are limited and could be easily improved by adding more staining at support of what claimed. An osteoclastic staining would be interesting as well and would increase the significance of the content.
A: Thank you for your comment. As you pointed out, if we performed long-term animal experiments, we had to observe the osteoclast dynamics in bone remodeling. However, the purpose of this experiment was to compare and observe the new bone formation on the titanium surface at the early stage. Therefore, we chose Villanueva-Bourne stain which can stain osteoid.
4) The abstract should be re-written to be more clear and easy to read. Minor rewriting would be also beneficial in the rest of the paper.
A: Thank you for pointing out.
We rewrote the abstract easier and easier to understand.
Reviewer 2 Report
UV treatment can enhance the surface energy of titanium that increase affinity with cell protein. However cleaning surface doesn't have same effect as UV. There was similar study and product which include ca ion in solution form. It will eventually make a connection with ca ion bridge between bone and titanium surface, which is less favorable outcome. I would like to know if S100 may cause any bridge connection between bone and titanium. Biochemical explannation should be included in this article about this type of connection. UV device is not that expensive now and costing time is only 15 minutes. I don't know if S100 effect is still better than UV.
Author Response
UV treatment can enhance the surface energy of titanium that increase affinity with cell protein. However cleaning surface doesn't have same effect as UV. There was similar study and product which include ca ion in solution form. It will eventually make a connection with ca ion bridge between bone and titanium surface, which is less favorable outcome. I would like to know if S100 may cause any bridge connection between bone and titanium. Biochemical explannation should be included in this article about this type of connection. UV device is not that expensive now and costing time is only 15 minutes. I don't know if S100 effect is still better than UV.
A: Thank you for pointing out. New UV irradiation equipment is now cheaper compared to the past. However, from this result S-100® does not need such special equipment, it proved that biological aging could be improved only by immersion for only 3 minutes, consider that there is its usefulness.
On the other hand, it has not been demonstrated whether S-100® directly engages Ca2+ ions to enhance titanium - bone integration. However, S-100® is a material with a negative charge rich in OH- ions, which can be inferred that polarity is imparted to the titanium surface and that Ca2+ ions are likely to bond.
Reviewer 3 Report
Comments
The study of “In Vitro and in Vivo Evaluation of Titanium Surface Modification for Biological Aging by Electrolytic Reducing Ionic Water” used electrolytic reducing ionic water to modify the properties of the titanium surface included hydrophilic ability, protein adsorption, cell biocompatibility, and new bone formation (in the animal study). Overall, it is an interesting and orientated study. I have only three questions.
1. In the fourth paragraph of the introduction section (line 60-65), authors used the word of “We” to introduce the matter of reference #10; however, in the reference # 10 the topic is related to the comparison between the chemically modified (SLActive®) and conventional SLA titanium implants and the authors in the article of the reference #10 is different from the authors in this manuscript. Please clarify it.
2. Sometimes, “changing the original titanium surface microstructure” is not a bad thing especially for the surfaces of dental implant and orthopedic joints. Since we already know that on a specific size of roughness implant surface the osseointegration can be reached faster.
3. In the surgical process of dental implant, the dental implant is screwed into the bone and the surface of the dental implant might be damaged slightly due to the shear and frictional forces in the surface. In the original technique, a new titanium oxide layer with an about 90-nm thickness can be formed and maybe after the surgical process of dental implant, those new titanium oxide layer can still be leave in the implant surface and be touched with bone to achieve its function (e.g. hydrophilic ability). However, by the new technique although it is easier to be used, after the surgical process of dental implant, how many “thickness” of this new modified titanium surface can be remained to achieve its function? I think that most of the readers who read this manuscript will have this kind of concern. Authors may need to provide more description to explain it.
Author Response
1) In the fourth paragraph of the introduction section (line 60-65), authors used the word of “We” to introduce the matter of reference #10; however, in the reference # 10 the topic is related to the comparison between the chemically modified (SLActive®) and conventional SLA titanium implants and the authors in the article of the reference #10 is different from the authors in this manuscript. Please clarify it.
A: As you pointed out, we corrected it because there was an error in quotation.
2) Sometimes, “changing the original titanium surface microstructure” is not a bad thing especially for the surfaces of dental implant and orthopedic joints. Since we already know that on a specific size of roughness implant surface the osseointegration can be reached faster.
A: Thank you for your comment. As you pointed out, it is possible to speed up osseointegration by giving a micro structure to the titanium surface. Therefore, since most of the dental implants already have a micro structure, we think that it is unnecessary to further change their fine structure. In this study, S-100 treatment was evaluated as a surface treatment method which can easily refresh biological aged titanium surface and improve bioactivity.
3) In the surgical process of dental implant, the dental implant is screwed into the bone and the surface of the dental implant might be damaged slightly due to the shear and frictional forces in the surface. In the original technique, a new titanium oxide layer with an about 90-nm thickness can be formed and maybe after the surgical process of dental implant, those new titanium oxide layer can still be leave in the implant surface and be touched with bone to achieve its function (e.g. hydrophilic ability). However, by the new technique although it is easier to be used, after the surgical process of dental implant, how many “thickness” of this new modified titanium surface can be remained to achieve its function? I think that most of the readers who read this manuscript will have this kind of concern. Authors may need to provide more description to explain it.
A: This S - 100 treatment does not change the structure of the titanium surface layer and the thickness of the titanium oxide layer does not change either.
The purpose of this treatment method is to increase the protein adsorption and cell adhesion by restoring the decreased hydrophilicity by biological aging without changing the surface structure in order to promote bone formation at the early stage after implantation.
We added this information in introduction.
Reviewer 4 Report
The authors attempt to achieve a surface modification of Ti by means of a biological surface modification agin by electrolitic reduction in ionic wáter.
The authors compare in vitro the titanium, and the modified material called S-100 and a material treated with UV. There is no comparison in vivo with UV. Why?
I understand the comparison between titanium and modified titanium S-100 both in vitro and in vivo. I do not see the need to compare only in vitro with the material treated with UV. Which is the reason? The authors indicate in the introduction (line 58-59) that there are already in vivo results of UV-treated titanium. Please include at the end of the introduction the reason why authors study only in vitro the UV material and with is the point to compare with S-100. It is no clear.
Abstract: first sentence: A titanium ....... osseointegration. It is a phrase of the introduction and not a result or an objective of the paper. Please, delete it.
M & M: It is complicated to understand the nomenclature of the samples. In the figures are given “fresh”, but on lines 73-79 are given “1 week and 4 weeks.” How is the S-100 fresh and the UV-fresh material made? I understand that the materials are 1 week + 3 minutes with S-100, and 4 weeks + 3 minutes with s-100 and the same for those treated with UV. And fresh material, how are they made? After 1 week or after 4 weeks? It is not clear, please rewrite.
Please always compare all obtained materials. There are no results of 1 week in figure 6 of protein adsorption, neither in figure 7 of cell adhesion, nor cellular attachment (fig 8).
Figure 9 does not indicate if the S-100 is after 1 week or 4 weeks.
Why is the point of do 1 week and 4 weeks if there is no comparison results?
Please include these results in the figures and the comments in the text.
If only one of the materials of the S-100 has been implanted, why two materials S-100 ( 1 week and 4 week) have been made? Based on the results with cells? Please explain it.
Line 122 indicate which of the S-100 materials has been implemented: 1 week + 3 minutes with S-100 or 4 weeks + 3 minutes with s-100.
Remove figure 1. It does not provide information relevant to the text. Also, there is not referenced in the text.
Figure caption 2. The blue line is not dop is continuous. Please change it.
Author Response
1) The authors attempt to achieve a surface modification of Ti by means of a biological surface modification agin by electrolitic reduction in ionic wáter.
The authors compare in vitro the titanium, and the modified material called S-100 and a material treated with UV. There is no comparison in vivo with UV. Why?
I understand the comparison between titanium and modified titanium S-100 both in vitro and in vivo. I do not see the need to compare only in vitro with the material treated with UV. Which is the reason? The authors indicate in the introduction (line 58-59) that there are already in vivo results of UV-treated titanium. Please include at the end of the introduction the reason why authors study only in vitro the UV material and with is the point to compare with S-100. It is no clear.
A: Thank you for your comment. As you pointed out, the effects of UV treatment has been already recognized in previous in vitro and in vivo studies. In this study, we compared the bioactive effects of S-100 as a new alternative surface treatment method and UV treatment as positive control. As a result, UV treatment showed significantly higher bioactivity than S - 100 treatment. Even though UV treated disks were used for in vivo study, the BIC of UV treated disk was considered to be significantly higher than S-100 treatment. Given the reduction in the number of animals, UV treated titanium disks were not used for in this in vivo study. Furthermore, as we received recommendations from our animal care committee to reduce the number of animals as much as possible according to the concept of 3R ("Replacement", "Reduction", "Refinement"), we excluded the UV treatment groups.
We added these contents to discussion.
2) Abstract: first sentence: A titanium ....... osseointegration. It is a phrase of the introduction and not a result or an objective of the paper. Please, delete it.
A: Following your suggestion, we deleted it.
3) M & M: It is complicated to understand the nomenclature of the samples. In the figures are given “fresh”, but on lines 73-79 are given “1 week and 4 weeks.” How is the S-100 fresh and the UV-fresh material made? I understand that the materials are 1 week + 3 minutes with S-100, and 4 weeks + 3 minutes with s-100 and the same for those treated with UV. And fresh material, how are they made? After 1 week or after 4 weeks? It is not clear, please rewrite.
A: Thank you for pointing out. The definition of “fresh” added to M&M.
4) Please always compare all obtained materials. There are no results of 1 week in figure 6 of protein adsorption, neither in figure 7 of cell adhesion, nor cellular attachment (fig 8).
Figure 9 does not indicate if the S-100 is after 1 week or 4 weeks.
Why is the point of do 1 week and 4 weeks if there is no comparison results?
Please include these results in the figures and the comments in the text.
A: Thank you for your comments. From the contact angle measurement results, it was confirmed that there was no significant difference between 1 week and 4 weeks. Therefore, we focused on comparison between fresh and 4 weeks on proteins assay and cell adhesion tests.
On the other hand, all cell proliferation tests were performed on the fresh dsks not for 1 or 4-week-aged disk at 24h and 72h.
5) If only one of the materials of the S-100 has been implanted, why two materials S-100 (1 week and 4 week) have been made? Based on the results with cells? Please explain it.
Line 122 indicate which of the S-100 materials has been implemented: 1 week + 3 minutes with S-100 or 4 weeks + 3 minutes with s-100.
A: In this study, we considered that the implantation period of 4 weeks is necessary to observe newly bone formation in vivo.
In addition, we used only 4 week-aged Ti disks for in vivo test. 4 week-aged Ti disks were immersed in S-100 for 3 minutes before implantation. Similarly, 4 week-aged Ti disks as Control were immersed in saline for 3 minutes.
6) Remove figure 1. It does not provide information relevant to the text. Also, there is not referenced in the text.
A: Since it is described in text lines 120 to 123, please confirm.
7) Figure caption 2. The blue line is not dop is continuous. Please change it.
A: Thank you for pointing out. We changed the caption from "blue dot" to "blue line".
Round 2
Reviewer 1 Report
After the changes made to the manuscript and the answer provided to the questions I think the manuscript is ready to be published in the present form
Reviewer 4 Report
I have carefully read the revised paper and believe that it is somewhat better than the previous submission. The critiques addressed by the reviewer were answered for the most part satisfactorily. The authors of the paper have clearly done a lot of work, and the results obtained by them are quite interesting. It reads better now.
